CD44 [2]

Angstrom6 has an IC50 of 10–100 nM, which effectively stops the migration of OVCAR8, OVCAR3, ES2, IGROV-1, MDA-MB-468, and MDA-MB361 cells [3]. As demonstrated by focal adhesion induction and MAP/ERK disrupting phosphorylation, Angstrom6 increases CD44-dependent interaction with hyaluronic acid and stimulates CD44-mediated signaling [3].

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Angstrom6 inhibited chemotaxis in a concentration-dependent manner in multiple human breast and ovarian cancer cell lines, with IC50 values of 10-100 nmol/L for responsive cell lines, suggesting physiological relevance. [2]
In Boyden chamber assays, Angstrom6 produced more than 85% inhibition of migration in CD44-positive SKOV3 ovarian cancer cells compared to untreated control, whereas it had no effect on migration of CD44-negative A2780 cells. [2]
Angstrom6 interfered with the binding of only one (DF1485) of four anti-CD44 antibodies tested, but did not interfere with binding of the anti-CD44 antibody IM7 (which blocks HA binding to CD44), indicating a subtle conformational change rather than global nonspecific change. [2]
Cross-linking and immunoprecipitation studies showed that Angstrom6 binds directly to CD44 on human ovarian SKOV3 cells. [2]
Angstrom6 modulated FAK phosphorylation in CD44-positive SKOV3 cells but not in CD44-negative A2780 cells, and this modulation was blocked by hyaluronan (HA), establishing a functional relationship between A6 and CD44-mediated intracellular signaling. [2]
In B16-F10 melanoma cells (which express CD44), Angstrom6 inhibited chemotaxis with an IC50 of 29 nmol/L. [2]
Angstrom6 increased the binding of CD44-expressing SKOV3 cells to HA-coated plates, an effect blocked by anti-CD44 antibody IM7, while having no effect on binding of CD44-non-expressing A2780 cells to HA-coated plates. [2]
Angstrom6 was shown to be directly cytotoxic for B-lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL) expressing ZAP-70, in a dose-dependent manner in vitro. [2]
Angstrom6 down-modulated expression of CD44 and ZAP-70, and inhibited B-cell receptor (BCR) signaling in human CLL B-cell lymphocytes. [2]
Angstrom6 did not demonstrate cytotoxicity in solid tumor models of glioma, breast, and ovarian cancer in vitro. [2]

Angstrom6 (100 mg/kg; subcutaneously twice daily) reduces the number of lung lesions produced by intravenous B16-F10 melanoma cells by 50% [3].

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In a mouse model of prostate cancer (orthotopic injection of human PC-3M-LN4 cells into BALB/c nu/nu mice), Angstrom6 treatment reduced the percentage of mice with lymph node metastases from >70% in control group to as low as 22%, and reduced lymph node volume by up to 70%. [2]
In a glioblastoma xenograft model (U87MG human glioma cells implanted subcutaneously in BALB/c nu/nu mice), Angstrom6 treatment suppressed tumor growth by 48% and prolonged time to progression after treatment discontinuation. Combination of Angstrom6 with cisplatin resulted in 92% inhibition of subcutaneous tumor growth and 98% inhibition in an intracranial model, and significantly increased survival time compared to either drug alone. [2]
In the B16-F10 lung metastatic model (C57BL/6 mice), Angstrom6 treatment reduced the number of lung metastases to 50% of control. [2]
In a mammary tumor model (MDA-MB-231 human mammary carcinoma xenografts in BALB/c nu/nu mice), Angstrom6 inhibited tumor growth by 90% compared to control and also inhibited metastasis. In Fisher rats inoculated with Mat B-III syngeneic mammary carcinoma cells, Angstrom6 inhibited tumor growth by 55% and markedly suppressed lymph node metastasis. Combination of Angstrom6 with tamoxifen resulted in 75% tumor growth inhibition. [2]
In a CLL xenograft model (ZAP-70-positive B-cell lymphocytes from patients injected into immunodeficient mice), Angstrom6 treatment resulted in up to 90% reduction in CLL burden. [2]
In a mouse model of laser-induced choroidal neovascularization (wet AMD), Angstrom6 treatment resulted in 95% inhibition of new vessel formation compared to non-treated control. [2]
In a rat model of laser-induced choroidal neovascularization, subcutaneous injections of Angstrom6 produced a 70% reduction in CNV compared to non-treated controls. [2]
In a primate model of laser-induced choroidal neovascularization, intravitreal administration of Angstrom6 resulted in a 71% reduction in CNV relative to control. [2]
In diabetic Brown Norway rats induced with streptozotocin, Angstrom6 treatment prevented loss of VE-cadherin and inhibited increase in microvascular permeability in the retina. [2]

Chemotaxis was evaluated using Boyden chamber assays. Cells were placed in the upper chamber, and chemoattractant in the lower chamber. Angstrom6 at various concentrations was added, and migrated cells were quantified after incubation. IC50 values were calculated for responsive cell lines. [2]
Flow cytometric analysis was performed to assess CD44 expression on cell lines using four different anti-CD44 antibodies. The effect of Angstrom6 on antibody binding was evaluated by pre-incubating cells with A6 before adding antibodies. [2]
For direct binding studies, human ovarian SKOV3 cells were bound and cross-linked to Angstrom6. Lysates of cross-linked cells were subjected to immunoprecipitation and immunoblotting to detect A6 binding to CD44. [2]
Intracellular signaling was studied by measuring FAK phosphorylation. CD44-positive SKOV3 and CD44-negative A2780 cells were treated with Angstrom6 in the presence or absence of hyaluronan, and cell lysates were analyzed by western blot using anti-phospho-FAK antibodies. [2]
Adhesion assays were performed by plating CD44-expressing SKOV3 or CD44-non-expressing A2780 cells onto HA-coated plates. Cells were pre-treated with Angstrom6 or anti-CD44 antibody IM7, and adherent cells were quantified. [2]
For CLL studies, B-lymphocytes from patients were isolated and cultured with increasing concentrations of Angstrom6. Cytotoxicity was measured, and expression of CD44 and ZAP-70 was assessed by flow cytometry. B-cell receptor signaling was evaluated by measuring downstream phosphorylation events. [2]

Animal/Disease Models: C57Bl/6 mice (carrying B16-F10 cells) [3]
Doses: 100 mg/kg
Route of Administration: Sc; twice (two times) daily for 11 days
Experimental Results: The number of pulmonary nodules was diminished, and the number of pulmonary metastases was diminished to control 50%.

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For prostate cancer model: human PC-3M-LN4 prostate cancer cells were injected orthotopically into the prostates of male BALB/c nu/nu mice. Angstrom6 was administered (dose and route not specified in this review) and lymph node metastases were measured. [2]
For glioblastoma models: U87MG human glioma cells were implanted subcutaneously or intracranially in BALB/c nu/nu mice. Animals were divided into treatment groups receiving Angstrom6 alone, cisplatin alone, or combination. Tumor growth and survival were monitored. [2]
For melanoma lung metastasis model: B16-F10 melanoma cells were injected into tail veins of C57BL/6 mice. Angstrom6 treatment was given, and lungs were evaluated for lesions at day 11. [2]
For mammary tumor models: MDA-MB-231 human mammary carcinoma cells were implanted subcutaneously in BALB/c nu/nu mice, or Mat B-III syngeneic mammary carcinoma cells were inoculated in Fisher rats. Angstrom6 was administered alone or with tamoxifen. Tumor growth and metastasis were assessed. [2]
For CLL xenograft model: ZAP-70-positive B-cell lymphocytes isolated from patients were injected into immunodeficient mice. Mice were treated with Angstrom6 or vehicle control, and CLL burden was evaluated. [2]
For choroidal neovascularization models: Laser-induced CNV was performed in mice, rats, and primates. Angstrom6 was administered subcutaneously (mice, rats) or intravitreally (primates). New vessel formation was quantified. [2]
For diabetic retinopathy model: Streptozotocin was used to induce diabetes in Brown Norway rats. Angstrom6 treatment was given, and retinal vascular permeability and VE-cadherin levels were measured. [2]

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For prostate cancer model: human PC-3M-LN4 prostate cancer cells were injected orthotopically into the prostates of male BALB/c nu/nu mice. Angstrom6 was administered (dose and route not specified in this review) and lymph node metastases were measured. [2]
For glioblastoma models: U87MG human glioma cells were implanted subcutaneously or intracranially in BALB/c nu/nu mice. Animals were divided into treatment groups receiving Angstrom6 alone, cisplatin alone, or combination. Tumor growth and survival were monitored. [2]
For melanoma lung metastasis model: B16-F10 melanoma cells were injected into tail veins of C57BL/6 mice. Angstrom6 treatment was given, and lungs were evaluated for lesions at day 11. [2]
For mammary tumor models: MDA-MB-231 human mammary carcinoma cells were implanted subcutaneously in BALB/c nu/nu mice, or Mat B-III syngeneic mammary carcinoma cells were inoculated in Fisher rats. Angstrom6 was administered alone or with tamoxifen. Tumor growth and metastasis were assessed. [2]
For CLL xenograft model: ZAP-70-positive B-cell lymphocytes isolated from patients were injected into immunodeficient mice. Mice were treated with Angstrom6 or vehicle control, and CLL burden was evaluated. [2]
For choroidal neovascularization models: Laser-induced CNV was performed in mice, rats, and primates. Angstrom6 was administered subcutaneously (mice, rats) or intravitreally (primates). New vessel formation was quantified. [2]
For diabetic retinopathy model: Streptozotocin was used to induce diabetes in Brown Norway rats. Angstrom6 treatment was given, and retinal vascular permeability and VE-cadherin levels were measured. [2]

In a Phase 1a clinical trial in normal volunteers, Angstrom6 administered subcutaneously at single dose levels of 150 and 300 mg/day showed a half-life (t1/2) of 1.8-2.0 hours at both dose levels. No cumulative increase in concentration over time was detected. Following subcutaneous administration twice daily for 6 days, no anti-A6 antibody production was detected at day 14. [2]

In animal studies, Angstrom6 showed no dose-limiting toxicity. [2]
In a Phase 1a double-blind, placebo-controlled trial in normal volunteers, there were no systemic drug-related adverse events. No significant alterations in physical examinations, vital signs, electrocardiograms, or clinical laboratory testing (including coagulation parameters PT, PTT, fibrinogen, thrombin time) were noted. [2]
In a Phase 1b trial in women with advanced gynecologic cancer, continuous treatment with Angstrom6 showed an excellent safety outcome with no specific toxicity profile. [2]
In a Phase 2 randomized, double-blind, placebo-controlled trial in ovarian cancer patients, the safety profile of Angstrom6 was comparable to that of placebo control. [2]

[1]. A phase 2, randomized, double-blind, placebo-controlled trial of clinical activity and safety of subcutaneous A6 in women with asymptomatic CA125 progression after first-line chemotherapy of epithelial ovarian cancer. Gynecol Oncol. 2008;111(1):89-94.

[2]. Finlayson M. Modulation of CD44 Activity by A6-Peptide. Front Immunol. 2015;6:135. Published 2015 Mar 30.

[3]. A6 peptide activates CD44 adhesive activity, induces FAK and MEK phosphorylation, and inhibits the migration and metastasis of CD44-expressing cells. Mol Cancer Ther. 2011;10(11):2072-2082.

Angstrom6 is an eight L-amino acid peptide (Ac-KPSSPPEE-NH2) derived from amino acid residues 136-143 of the connecting peptide domain of human urokinase plasminogen activator (uPA). It does not bind to the uPA receptor (uPAR) nor interfere with uPA/uPAR binding. [2]
Angstrom6 shares sequence homology with a portion of the link domain of CD44 (CD44 amino acid residues 120-NASAPPEE-127), a region conserved in all CD44 isoforms, straddling the CD44 splice junction of exons 3 and 4, including a potential glycosylation site, and involved in hyaluronan binding. [2]
Angstrom6 binds to CD44 resulting in inhibition of migration, invasion, and metastasis of tumor cells, and modulation of CD44-mediated cell signaling. It acts by inhibiting the metastatic process including extravasation and/or metastatic colonization. [2]
Angstrom6 has demonstrated efficacy and an excellent safety profile in Phase 1a, 1b, and 2 clinical trials for cancer. It has also shown promising results for treatment of diabetic retinopathy and wet age-related macular degeneration through reduction of retinal vascular permeability and inhibition of choroidal neovascularization. [2]
Angstrom6 may render chemoresistant tumor cells sensitive to chemotherapy, as shown in combination studies with tamoxifen (breast cancer) and cisplatin (glioma). Its superior safety profile (no immunogenicity, no dose-limiting toxicities, no serious side-effects) makes it suitable for long-term or maintenance therapy to prevent recurrence from micrometastases. [2]
The precise mechanism of Angstrom6 has not been fully defined, but evidence supports action through a CD44-mediated pathway, possibly by inducing conformational changes in CD44 or CD44 dimerization, or by influencing CD44 co-receptor activity (e.g., c-Met). [2]

C39H62N10O15

910.967589855194

910.44

220334-14-5

42638946

White to off-white solid powder

11

16

25

64

1750

8

O=C([C@@H]1CCCN1C([C@H](CO)NC([C@H](CO)NC([C@@H]1CCCN1C([C@H](CCCCN)NC(C)=O)=O)=O)=O)=O)N1CCC[C@H]1C(N[C@H](C(N[C@H](C(=O)O)CCC(N)=O)=O)CCC(=O)O)=O

NJSODKGEFNFGRI-PJYAFMLMSA-N

InChI=1S/C39H62N10O15/c1-21(52)42-24(7-2-3-15-40)37(62)47-16-4-8-27(47)36(61)45-25(19-50)34(59)46-26(20-51)38(63)49-18-6-10-29(49)39(64)48-17-5-9-28(48)35(60)44-23(12-14-31(55)56)33(58)43-22(32(41)57)11-13-30(53)54/h22-29,50-51H,2-20,40H2,1H3,(H2,41,57)(H,42,52)(H,43,58)(H,44,60)(H,45,61)(H,46,59)(H,53,54)(H,55,56)/t22-,23-,24-,25-,26-,27-,28-,29-/m0/s1

(4S)-4-[[(2S)-2-[[(2S)-1-[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-acetamido-6-aminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-4-carboxybutanoyl]amino]-5-amino-5-oxopentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~109.77 mM)
H2O : ≥ 100 mg/mL (~109.77 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)